A central issue in cancer chemotherapy is the severe toxic side effects of anticancer agents on healthy tissues, which invariably imposes dose reduction, treatment delay or even discontinuance of therapy (Fennelly (1995) Clin. Cancer Res. 1:575-582; Hanjani, et al. (2002) Gynecol. Oncol. 85:278-284; Kobayashi, et al. (2002) Chronobiol. Int. 19:237-251; Ross and Small (2002) J. Urol. 167:1952-1956; Markman, et al. (2002) J. Clin. Oncol. 20:2365-2369; Sehouli, et al. (2002) Gynecol. Oncol. 85:321-326). Cytotoxicity for healthy organs can be significantly diminished by employing a drug delivery system which targets cancer cells (Alvarez, et al. (2002) Expert. Opin. Biol. Ther. 2:409-417; Dass and Su (2001) Drug Deliv. 8:191-213; Kopecek, et al. (2001) J. Controlled Rel. 74:147-158; Kunath, et al. (2000) Eur. J. Pharm. Biopharm. 49:11-15; Minko, et al. (2001) Dis. Manag. Clin. Outcomes 3:48-54; Vasey, et al. (2002) J. Clin. Oncol. 20:1562-1569). The usage of these drug delivery systems prevents, in most cases, the uptake of the drug by normal cells and enhances the influx and retention of the drug in cancer cells.
A second factor that limits the success of chemotherapeutic treatment of ovarian carcinoma is the development of multidrug resistance (Fennelly (1995) supra). The term multidrug resistance (MDR) is used to describe the resistance against a broad spectrum of anticancer drugs after the treatment with a single agent. A membrane glycoprotein, termed P-glycoprotein, has been shown to be responsible for cross-resistance to a broad range of structurally and functionally distinct cytotoxic agents. P-glycoprotein, encoded in humans by the MDR1 gene, functions as an energy-dependent membrane pump to remove cytotoxic agents from the resistant cells (Szakacs, et al. (1998) Pathol. Oncol. Res. 4:251-257). In addition to P-glycoprotein, other transporters contributing to MDR of cancer cells, such as the multidrug resistance associated protein (MRP), have been identified (van Veen and Konings (1998) Biochem. Biophys. Acta 1365:31-36). The overexpression of genes encoding these drug efflux pumps and an increase in their activity are the main causes of pump resistance in human ovarian carcinoma (Minko, et al. (2001) supra; Hamaguchi, et al. (1993) Cancer Res. 53:5225-5232; Minko, et al. (1998) J. Controlled Rel. 54:223-233; Minko, et al. (1999) J. Controlled Rel. 59:133-148; Pakunlu, et al. (2003) Pharmaceut. Res. 20:351-359).
Several methods, including the use of antisense oligonucleotides targeted against mRNA encoded by genes of drug efflux pumps, were developed over the last decades to overcome or suppress multidrug resistance (Alahary, et al. (1998) JPET 286:419-428; Motomura, et al. (1998) Blood 91:3163-3171; Corrias and Tonini (1992) Anticancer Res. 12:1431-1438). While these compounds lead to an increase in intracellular drug concentration, they do not overcome the adaptive activation of cell death defense, also known as non-pump resistance (Minko, et al. (2001) supra). It is known that the up-regulation of the cellular antiapoptotic system plays a role in this second line of defense and BCL-2 family proteins are key proteins in this system (Gross, et al. (1999) Genes Dev. 13:1899-1911; Reed (1999) J. Clin. Oncol. 17:2941-2953). Unlike the drug efflux pump proteins, overexpression of BCL-2 protein does not interfere with the entry and accumulation of drugs in tumor cells. Instead, BCL-2 protein prevents drug-induced damage from being efficiently translated into cell death by preventing cytochrome c release from mitochondria which triggers the caspase cascade of apoptosis execution.
The BCL-2 protein family consists of two kinds of proteins with counter-modulating functions; a group that suppress apoptosis, if overexpressed, and a group that has the ability to induce apoptosis (Reed (1999) supra; Abate-Shen and Shen (2000) Genes Dev. 14:2410-2434; Lowe and Lin (2000) Carcinogenesis 21:485-495). Although the precise role of these proteins in apoptosis induction and development of resistance during cancer therapy remains unclear, it was found that the expression ratio of antiapoptotic members of BCL-2 protein family to proapoptotic members determines survival or death following an apoptotic stimulus (Oltvai, et al. (1993) Cell 74:609-619) Several studies have correlated the expression of BCL-2 family members with a survival advantage in ovarian cancer but failed to find an association with overall response to chemotherapy (Baekelandt, et al. (1999) Clin. Oncol. 17:2061; Herod, et al. (1996) Cancer Res. 56:2178-2184; Schuyer, et al. (2001) Br. J. Cancer 85:1359-1367). In contrast, BCL-2 overexpression has been reported to be associated with a poor prognosis and resistance to chemotherapy (Kassim, et al. (1999) Clin. Biochem. 32:333-338; Mano, et al. (1999) Eur. J. Cancer 35:1214-1219). These differences may be explained by the fact that clinical studies focus on the separate analysis of the expression of pro- or anti-apoptotic members of the BCL-2 protein family. Concurrently, it was shown that it is the ratio between the expression of anti- and proapoptotic proteins that determines cell death by apoptosis after chemotherapy (Reed (1999) supra; Oltvai, et al. (1993) supra; Schuyer, et al. (2001) supra).
The BCL-2 family is characterized by specific regions of homology termed BCL-2 homology (BH1, BH2, BH3, BH4) domains. These domains are critical to the function of these proteins, including their impact on cell survival and their ability to interact with other family members and regulatory proteins (Abate-Shen and Shen (2000) supra; Johnson (1999) Endocrinology 140:5465-5467). It was found that the BH3 domain of proapoptotic proteins from the BCL-2 family is responsible for the induction of apoptosis (Abate-Shen and Shen (2000) supra; Johnson (1999) Endocrinology 140:5465-5467; Cosulich, et al. (1997) Curr. Biol. 7:913-920). Furthermore, expression of small, truncated derivatives of the BAK protein containing the BH3 domain are sufficient for cell killing activity (Lutz (2000) Biochem. Sci. Trans. 28:51-56). Moreover, it was found that short synthetic peptides, corresponding to the minimal sequence of BH3 domain when bound to the antiapoptotic BCL-2 family proteins, suppress the cellular antiapoptotic defense (Minko, et al. (2001) supra; Lutz (2000) supra; Holinger, et al. (1999) J. Biol. Chem. 274:13298-13304; Minko, et al. (2002) Cancer Chemother. Pharmacol. 50:143-150). While, BH3 peptide may potentially improve traditional therapy of ovarian cancer by decreasing the resistance of cancer cells to chemotherapeutic agents, the practical use of the BH3 peptide is limited by its low permeation into cancer cells.
A targeted approach for producing a net increase in apoptosis induction during treatment of cancer to significantly increase cancer cell death and efficacy of chemotherapy is needed. The present invention meets this long-felt need.